登革病毒HLA-A*0201限制性T细胞表位诱导免疫反应的研究

Study on the ability of dengue virus HLA-A*0201-restricted T-cell epitopes in inducing T cell response

目的:探讨登革病毒(DENV)HLA-A*0201限制性T细胞表位诱导DENV血清型间交叉反应性T细胞的效应。方法:基于已鉴定DENV-1中HLA-A*0201限制性CD8+T细胞表位(NS4b40-48TLYAVATTI)序列,合成在其他血清型中相似的候选表位,采用MHC-肽复合物稳定性实验检测候选表位与HLA-A*0201结合力。采用酶联免疫斑点(ELISPOT)实验研究已鉴定表位及候选表位免疫HLA-A*0201转基因小鼠产生的CD8+T细胞识别相同及相似表位能力;采用ELISPOT实验研究不同DENV血清型感染HLA-A*0201转基因小鼠是否产生能识别已鉴定表位及候选表位的T细胞反应。结果:DENV-3中NS4b39-47TLYAVATTV与HLA-A*0201具高结合力,但DENV-2中NS4b39-47TLYAVATTF与HLA-A*0201结合力弱。DENV-1-NS4b40-48和DENV-3-NS4b39-47免疫的转基因小鼠脾、淋巴结和外周血中存在可识别DENV-1-NS4b40-48、DENV-2-NS4b39-47和DENV-3-NS4b39-47的交叉反应性CD8+T细胞。在DENV-1,-2,-3感染的转基因小鼠体内也存在类似的T细胞反应。结论:DENV-3中NS4b39-47TLYAVATTV为新的CD8+T细胞表位,该表位及已报道表位NS4b40-48TLYAVATTI均可诱导DENV血清型间交叉反应性CD8+T细胞反应,二者有助于我们研究DENV血清型间交叉反应性CD8+T细胞的功能以及用于评估DENV候选疫苗的T细胞免疫效应。

Objective： To explore the ability of dengue virus （DENV）-specific HLA-A＊0201-restricted T cell-epitope to induce DENV serotype cross-reactive T cell response. Methods： Based on the sequence of one previously identified DENV-l-specific HLA-A＊0201-restricted CD8＋ T-cell epitope （NS4b40-48TLYAVATTI）, the epitope variants in other DENV serotypes were synthesized and their binding affinity for HLA-A＊0201 molecule was determined by using MHC-peptide complex stabilization assay. These peptides were used to immunize HLA-A＊0201 transgenic mice, and the ability of induced CD8＋T cells to recognize the same epitope and epi- tope variant was evaluated by using enzyme-linked immunospot （ELISPOT） assay; Finally, individual DENV serotype was used to infect HLA-A＊0201 transgenic mice and ELISPOT assay was utilized to assess whether the induced CD8＋ T cells could recognize same epitope or epitope variant. Results： The epitope candidate in DENV-3 （NS4b39.47TLYAVATTV） had high affinity for HLA-A＊0201 molecule while the epitope candidate in DENV-2 （NS4b39.47TLYAVATTF） bound to HLA-A＊0201 molecule with a low affinity. Both DENV-1-NS4b40_48 and DENV-3-NS4b39_47 elicited DENV serotype cross-reactive CD8＋ T cells which were distributed in the spleen, lymph nodes, peripheral blood of peptide-immunized transgenic mice and could recognize DENV-1-NS4b40_48, DENV-2-NS4b39_47 and DENV-3-NS4b39_47 simultaneously. Additionally, the same phenomenon was observed in DENV-1, -2, -3-infected HLA-A＊0201 transgenic mice. Conclusion： DENV-3-derived NS4b39.47TLYAVATTV is identified as novel CD8＋ T-cell epitope. Both this epitope and a previously identified epitope （NS4b40.48TLY- AVATTI） triggered DENV serotype cross-reactive CD8＋ T cell response, which would help us clarify the ftmction of DENV serotype cross-reactive CD8＋ T cells and evaluate the immunogenicity of DENV vaccine.